Crystalline forms of lnt

ABSTRACT

The invention relates to crystalline polymorphs of lacto-N-tetraose (LNT) and methods for making the same for use in pharmaceutical compositions, nutritional formulations and food supplements.

FIELD OF THE INVENTION

The present invention provides crystalline modifications ofGalpβ1-3GlcNAcpβ1-3Galpβ1-4Glc (lacto-N-tetraose, LNT), a method ofmaking them and formulations containing the same.

BACKGROUND OF THE INVENTION

The human milk oligosaccharide LNT has previously been crystallized.Kuhn et al. [Chem. Ber. 86, 827, (1953)] isolated LNT from mother's milkfor the first time and crystallized it from aqueous ethanol. Upondrying, the air-dried crystalline material lost more than 10% of itsweight (melting point 200-205° C. (dec.)), but its crystal structure didnot change according to X-ray power diffraction measurements. Theso-dried material proved to be hygroscopic as it took back around 8% ofits weight when stored in air. Later, the same authors reported theobtention of crystalline LNT by partial hydrolysis oflacto-N-fucopentaose I [Chem. Ber. 89, 2514 (1956)] or sialylated LNT[Chem. Ber. 95, 513 (1962)], both isolated from mother's milk, followedby crystallization; their reported decomposition points were 205±10° C.(“depending on water content and the speed of heating”) and 211-214° C.,respectively; both samples were considered to be identical with thepreviously reported one based on their X-ray power diffractogram and/orIR spectrum. Similarly, Malpress et al. [Biochem. J. 68, 708 (1958)]isolated LNT from mother's milk having a melting point of 204-205° C.Takamura et al. [Chem. Pharm. Bull. 27, 1497 (1979) and ibid. 28, 1804(1980)] reported a melting point of 225-228° C. for LNT made by chemicaltotal synthesis and crystallized from aqueous ethanol.

Crystallization or recrystallization is one of the simplest and cheapestmethods to separate a product from contaminants and obtain a puresubstance. Providing alternative crystalline forms of a solid compoundis an important aspect of the product's development, because thedifferent crystalline forms affect the compound's properties—forexample, thermodynamic stability, solubility, density, hygroscopicity,electrical properties (such as dielectric constant, conductivity),mechanical properties (such as friability, hardness, breaking strength,elasticity), optical properties (such as colour, transparency,refraction), etc.—diversely. Polymorphs or other crystalline formsprovide alternative means for improving a product's characteristics andusefulness.

For this reason, ways have been sought for obtaining other crystallineforms of LNT which have different, preferably advantageous, physicalproperties (such as e.g. hygroscopicity, stability, dissolution rate,purity, filterability, drying properties, flowability of the powder,etc.) for facilitating its isolation from a complex milieu or its use inpharmaceutical or nutritional compositions.

SHORT DESCRIPTION OF THE INVENTION

The present invention provides novel crystalline polymorphs of LNT,methods for making the novel crystalline polymorphs of LNT that can beproduced in large scale. Thus, the crystalline products of thisinvention are each a high purity LNT that are suitable for nutritionaland pharmaceutical products.

BRIEF DESCRIPTION OF THE FIGURES

The invention will be described in further detail hereinafter withreference to the accompanying figures, in which:

FIG. 1 shows the X-ray powder diffraction pattern of the crystalline LNTform A;

FIG. 2 shows the X-ray powder diffraction pattern of the crystalline LNTform B;

FIG. 3 shows the X-ray powder diffraction pattern of the crystalline LNTform C;

FIG. 4 shows the X-ray powder diffraction pattern of the crystalline LNTform D.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a first LNT crystalline form A. The crystallineLNT form A comprises X-ray powder diffraction reflections, based on ameasurement using CuKα radiation, at 19.21±0.2 2Θ angle, preferably at19.21 and 4.20±0.2 2Θ angles, more preferably at 19.21, 4.20 and19.99±0.2 2Θ angles, yet more preferably at 19.21, 4.20, 19.99 and20.63±0.2 2Θ angles, even more preferably at 19.21, 4.20, 19.99, 20.63and 18.80±0.2 2Θ angles, even more preferably at 19.21, 4.20, 19.99,20.63, 18.80 and 19.82±0.2 2Θ angles, even more preferably at 19.21,4.20, 19.99, 20.63, 18.80, 19.82 and 8.60±0.2 2Θ angles, even morepreferably at 19.21, 4.20, 19.99, 20.63, 18.80, 19.82, 8.60 and 6.52±0.22Θ angles, most preferably at 19.21, 4.20, 19.99, 20.63, 18.80, 19.82,8.60, 6.52 and 20.47±0.2 2Θ angles, particularly at 19.21, 4.20, 19.99,20.63, 18.80, 19.82, 8.60, 6.52, 20.47 and 21.96±0.2 2Θ angles.

In one embodiment, the crystalline LNT form A comprises X-ray powderdiffraction reflections, based on a measurement using CuKα radiation, at19.21±0.1 2Θ angle, preferably at 19.21 and 4.20±0.1 2Θ angles, morepreferably at 19.21, 4.20 and 19.99±0.1 2Θ angles, yet more preferablyat 19.21, 4.20, 19.99 and 20.63±0.1 2Θ angles, even more preferably at19.21, 4.20, 19.99, 20.63 and 18.80±0.1 2Θ angles, even more preferablyat 19.21, 4.20, 19.99, 20.63, 18.80 and 19.82±0.1 2Θ angles, even morepreferably at 19.21, 4.20, 19.99, 20.63, 18.80, 19.82 and 8.60±0.1 2Θangles, even more preferably at 19.21, 4.20, 19.99, 20.63, 18.80, 19.82,8.60 and 6.52±0.1 2Θ angles, most preferably at 19.21, 4.20, 19.99,20.63, 18.80, 19.82, 8.60, 6.52 and 20.47±0.1 2Θ angles, particularly at19.21, 4.20, 19.99, 20.63, 18.80, 19.82, 8.60, 6.52, 20.47 and 21.96±0.12Θ angles.

In other embodiment, the crystalline LNT form A has an X-ray powderdiffraction diagram as shown in FIG. 1.

List of peaks of the XRPD pattern of the crystalline LNT form A isreported in Table 1.

TABLE 1 2Θ rel. intensity 4.20 80 6.22 15 6.52 25 7.04 16 8.60 25 10.148 10.61 4 11.11 18 12.47 5 12.62 12 13.09 8 13.32 5 13.40 5 13.68 414.38 3 14.80 5 16.34 10 16.52 22 17.21 10 18.26 11 18.80 43 19.21 10019.76 20 19.82 30 19.99 55 20.16 20 20.35 10 20.47 25 20.63 45 21.32 2021.96 25

LNT crystalline form A has a capillary melting point of 226-231° C. Theconventionally dried sample (in desiccator over P₂O₅) has a watercontent of 12.5-13.0 wt %. In TG-DSC measurement a weight loss of about5.3 wt % is observed between 35 and 120° C.

This invention also provides a second LNT crystalline form B. Thecrystalline LNT form B comprises X-ray powder diffraction reflections,based on a measurement using CuKα radiation, at 19.25±0.2 2Θ angle,preferably at 19.25 and 9.19±0.2 2Θ angles, more preferably at 19.25,9.19 and 6.88±0.2 2Θ angles, yet more preferably at 19.25, 9.19, 6.88and 21.07±0.2 2Θ angles, even more preferably at 19.25, 9.19, 6.88,21.07 and 20.87±0.2 2Θ angles, even more preferably at 19.25, 9.19,6.88, 21.07, 20.87 and 19.94±0.2 2Θ angles, even more preferably at19.25, 9.19, 6.88, 21.07, 20.87, 19.94 and 19.66±0.2 2Θ angles, evenmore preferably at 19.25, 9.19, 6.88, 21.07, 20.87, 19.94, 19.66 and6.02±0.2 2Θ angles, most preferably at 19.25, 9.19, 6.88, 21.07, 20.87,19.94, 19.66, 6.02 and 22.30±0.2 2Θ angles, particularly at 19.25, 9.19,6.88, 21.07, 20.87, 19.94, 19.66, 6.02, 22.30 and 25.15±0.2 2Θ angles.

In one embodiment, the crystalline LNT form B comprises X-ray powderdiffraction reflections, based on a measurement using CuKα radiation, at19.25±0.1 2Θ angle, preferably at 19.25 and 9.19±0.1 2Θ angles, morepreferably at 19.25, 9.19 and 6.88±0.1 2Θ angles, yet more preferably at19.25, 9.19, 6.88 and 21.07±0.1 2Θ angles, even more preferably at19.25, 9.19, 6.88, 21.07 and 20.87±0.1 2Θ angles, even more preferablyat 19.25, 9.19, 6.88, 21.07, 20.87 and 19.94±0.1 2Θ angles, even morepreferably at 19.25, 9.19, 6.88, 21.07, 20.87, 19.94 and 19.66±0.1 2Θangles, even more preferably at 19.25, 9.19, 6.88, 21.07, 20.87, 19.94,19.66 and 6.02±0.1 2Θ angles, most preferably at 19.25, 9.19, 6.88,21.07, 20.87, 19.94, 19.66, 6.02 and 22.30±0.1 2Θ angles, particularlyat 19.25, 9.19, 6.88, 21.07, 20.87, 19.94, 19.66, 6.02, 22.30 and25.15±0.1 2Θ angles. In other embodiment, the crystalline LNT form B hasan X-ray powder diffraction diagram as shown in FIG. 2.

List of peaks of the XRPD pattern of the crystalline LNT form A isreported in Table 2.

TABLE 2 2Θ rel. intensity 6.02 25 6.88 78 9.10 10 9.19 80 9.63 3 9.70 1010.73 12 12.05 1 13.78 18 15.43 8 17.49 3.5 17.55 4 18.11 13 18.40 519.25 100 19.26 1 19.66 25 19.94 31 20.83 1 20.87 35 21.07 40 21.55 821.67 10 22.30 22 22.95 8 23.30 19 23.40 15 23.94 6 24.12 5 24.23 1325.15 19 25.29 15 26.15 3.5 26.35 6.5 27.12 13 27.29 9 27.52 8 28.06 528.11 10 28.50 11 28.68 6

LNT crystalline form B has a capillary melting point 239-243° C. The airdried sample has a water content of 8.9-9.2 wt %.

This invention also provides a third LNT crystalline form C. Thecrystalline LNT form C comprises X-ray powder diffraction reflections,based on a measurement using CuKα radiation, at 20.22±0.2 2Θ angle,preferably at 20.22 and 9.36±0.2 2Θ angles, more preferably at 20.22,9.36 and 19.80±0.2 2Θ angles, yet more preferably at 20.22, 9.36, 19.80and 20.47±0.2 2Θ angles, even more preferably at 20.22, 9.36, 19.80,20.47 and 4.67±0.2 2Θ angles, even more preferably at 20.22, 9.36,19.80, 20.47, 4.67 and 18.79±0.2 2Θ angles, even more preferably at20.22, 9.36, 19.80, 20.47, 4.67, 18.79 and 6.82±0.2 2Θ angles, even morepreferably at 20.22, 9.36, 19.80, 20.47, 4.67, 18.79, 6.82 and 21.82±0.22Θ angles, even more preferably at 20.22, 9.36, 19.80, 20.47, 4.67,18.79, 6.82, 21.82 and 7.93±0.2 2Θ angles, most preferably at 20.22,9.36, 19.80, 20.47, 4.67, 18.79, 6.82, 21.82, 7.93 and 19.16±0.2 2Θangles, particularly 20.22, 9.36, 19.80, 20.47, 4.67, 18.79, 6.82,21.82, 7.93, 19.16 and 19.45±0.2 2Θ angles.

In one embodiment, the crystalline LNT form C comprises X-ray powderdiffraction reflections, based on a measurement using CuKα radiation, at20.22±0.1 2Θ angle, preferably at 20.22 and 9.36±0.1 2Θ angles, morepreferably at 20.22, 9.36 and 19.80±0.1 2Θ angles, yet more preferablyat 20.22, 9.36, 19.80 and 20.47±0.1 2Θ angles, even more preferably at20.22, 9.36, 19.80, 20.47 and 4.67±0.1 2Θ angles, even more preferablyat 20.22, 9.36, 19.80, 20.47, 4.67 and 18.79±0.1 2Θ angles, even morepreferably at 20.22, 9.36, 19.80, 20.47, 4.67, 18.79 and 6.82±0.1 2Θangles, even more preferably at 20.22, 9.36, 19.80, 20.47, 4.67, 18.79,6.82 and 21.82±0.1 2Θ angles, even more preferably at 20.22, 9.36,19.80, 20.47, 4.67, 18.79, 6.82, 21.82 and 7.93±0.1 2Θ angles, mostpreferably at 20.22, 9.36, 19.80, 20.47, 4.67, 18.79, 6.82, 21.82, 7.93and 19.16±0.1 2Θ angles, particularly 20.22, 9.36, 19.80, 20.47, 4.67,18.79, 6.82, 21.82, 7.93, 19.16 and 19.45±0.1 2Θ angles.

In other embodiment, the crystalline LNT form C has an X-ray powderdiffraction diagram as shown in FIG. 3.

List of peaks of the XRPD pattern of the crystalline LNT form C isreported in Table 3.

TABLE 3 2Θ rel. intensity 4.67 50 6.82 42 7.39 25 7.93 32 9.36 75 11.3510 11.46 16 12.06 4 12.49 15 13.66 8 14.83 5 15.09 15 15.89 4 17.15 2118.18 7 18.79 48 19.16 30 19.42 20 19.45 30 19.80 55 20.01 5 20.22 10020.47 52 20.84 15 21.82 34 21.85 10 22.20 20 22.45 25 23.07 4 23.36 723.83 8 23.85 15 23.93 10 24.53 4 24.60 3 24.72 3 25.14 6 25.38 11 25.456 26.10 5 26.54 10 26.620 5 26.71 15 27.80 10

LNT crystalline form C has a capillary melting point 249-255° C. The airdried sample has a water content of 6.4-9.4 wt %. Indexing of the XRPDdiagram of form C results in the following cell parameters:

cell parameters symmetry space group a b c α β γ cell volumeorthorhombic P212121 23.93 Å 30.85 Å 4.778 Å 90 90 90 3527.6 Å³

In one embodiment, LNT crystalline form C can be prepared in thefollowing way: LNT is dissolved in around 2.5-3.5 volumes of water andheated to around 55-65° C. Methanol (around 6-7.5 volumes) is thenslowly added to aqueous solution. The so-obtained solution is stirred atthe same temperature until spontaneous nucleation, then the suspensionis slowly cooled to 0-25° C. and the crystals of LNT polymorph C areseparated by filtration.

Alternatively, a 20-25 w/v % LNT solution in water is heated to around30-40° C. to which, under stirring, methanol is added in two steps: a)first around 4-5 volumes of methanol to give a clear solution, then b)the same volumes of methanol in portions, preferably in around 0.6-0.85volumes portions, to give a suspension. In some embodiments, preferably,between step a) and b) seeding crystals of LNT polymorph C can be added.After completion of the methanol addition the suspension is stirredbetween 30-60° C. for a couple of hours, then slowly cooled to 0-25° C.and the crystals of LNT pdymorph C are separated by filtration.

In the above crystallization methods, the term “around” means adeviation from the indicated value by 5-10%; and the term “volume” meansthat how many ml of solvent is added to 1 g of LNT.

After LNT form C is crystallized (by any means), the crystals can bedried in vacuum for less than one day, typically less than 21 hours at30-50 mbar and at not more than 50-60° C. In case of drying atatmospheric pressure, the temperature can be as high as 70° C., but thedrying time preferably does not exceed 5 hours. At lower temperatureproportionally longer drying time can be applied.

This invention also provides a fourth LNT crystalline form D. Thecrystalline LNT form D comprises X-ray powder diffraction reflections,based on a measurement using CuKα radiation, at 19.17±0.2 2Θ angle,preferably at 19.17 and 20.91±0.2 2Θ angles, more preferably at 19.17,20.91 and 10.67±0.2 2Θ angles, yet more preferably at 19.17, 20.91,10.67 and 15.90±0.2 2Θ angles, even more preferably at 19.17, 20.91,10.67, 15.90 and 9.32±0.2 2Θ angles, even more preferably at 19.17,20.91, 10.67, 15.90, 9.32 and 21.16±0.2 2Θ angles, even more preferablyat 19.17, 20.91, 10.67, 15.90, 9.32, 21.16 and 23.44±0.2 2Θ angles, evenmore preferably at 19.17, 20.91, 10.67, 15.90, 9.32, 21.16, 23.44 and21.40±0.2 2Θ angles, even more preferably at 19.17, 20.91, 10.67, 15.90,9.32, 21.16, 23.44, 21.40 and 8.99±0.2 2Θ angles, most preferably at19.17, 20.91, 10.67, 15.90, 9.32, 21.16, 23.44, 21.40, 8.99 and15.00±0.2 2Θ angles, particularly 19.17, 20.91, 10.67, 15.90, 9.32,21.16, 23.44, 21.40, 8.99, 15.00 and 20.64±0.2 2Θ angles.

In one embodiment, the crystalline LNT form D comprises X-ray powderdiffraction reflections, based on a measurement using CuKα radiation, at19.17±0.1 2Θ angle, preferably at 19.17 and 20.91±0.1 2Θ angles, morepreferably at 19.17, 20.91 and 10.67±0.1 2Θ angles, yet more preferablyat 19.17, 20.91, 10.67 and 15.90±0.1 2Θ angles, even more preferably at19.17, 20.91, 10.67, 15.90 and 9.32±0.1 2Θ angles, even more preferablyat 19.17, 20.91, 10.67, 15.90, 9.32 and 21.16±0.1 2Θ angles, even morepreferably at 19.17, 20.91, 10.67, 15.90, 9.32, 21.16 and 23.44±0.1 2Θangles, even more preferably at 19.17, 20.91, 10.67, 15.90, 9.32, 21.16,23.44 and 21.40±0.1 2Θ angles, even more preferably at 19.17, 20.91,10.67, 15.90, 9.32, 21.16, 23.44, 21.40 and 8.99±0.1 2Θ angles, mostpreferably at 19.17, 20.91, 10.67, 15.90, 9.32, 21.16, 23.44, 21.40,8.99 and 15.00±0.1 2Θ angles, particularly 19.17, 20.91, 10.67, 15.90,9.32, 21.16, 23.44, 21.40, 8.99, 15.00 and 20.64±0.1 2Θ angles.

In other embodiment, the crystalline LNT form D has an X-ray powderdiffraction diagram as shown in FIG. 4.

List of peaks of the XRPD pattern of the crystalline LNT form D isreported in Table 4.

TABLE 4 2Θ rel. intensity 5.31 14 8.99 15 9.32 24 10.28 6 10.67 70 11.246 12.11 9 13.68 3 15.00 15 15.40 3 15.90 25 17.44 2 18.07 4 18.72 719.17 100 19.73 10 20.40 10 20.64 15 20.91 72 21.16 20 21.40 16 21.66 922.12 6 22.97 11 23.44 20 24.16 8 24.48 14 25.04 13 25.78 7 26.27 926.68 6 26.96 8 27.60 4 28.36 8 29.52 3 30.08 9 30.76 3 31.20 5 31.48 333.40 6 33.799 7 34.36 9 22.12 6

LNT crystalline form D has a capillary melting point 249-251° C. The airdried sample has a water content of about 2.7 wt %.

LNT crystalline form D can be typically obtained form C under forceddrying conditions so that form C undergoes dehydration while thecrystalline phase changes. For example the vacuum drying requiresseveral days to convert form C to form D. At atmospheric pressure, ifthe temperature is high, shorter time may be sufficient.

The powder X-ray diffraction patterns of the crystalline LNT forms A toD of this invention are different from each other as the notabledifferences show in their diffractogram.

Each of the LNT crystalline forms A, B, C and D can be considered as ananomeric mixture of α- and β-anomers or even a pure form of one of theanomers.

Preferably, each of the crystalline LNT forms A, B, C and D of thisinvention is substantially pure. The expression “substantially pure”preferably means that the crystalline LNT polymorph contains less than10 w/w % of impurity, preferably less than 5 w/w % of impurity, morepreferably less than 1 w/w % of impurity, most preferably less than 0.5w/w % of impurity, in particular less than 0.1 w/w % of impurity,wherein “impurity” refers to any physical entity different from thecrystalline LNT form in question, such as an amorphous LNT, differentLNT polymorph(s) or form(s), unreacted intermediate(s) remained from thesynthesis of LNT, by-product(s), degradation product(s), inorganicsalt(s) and/or other contaminants different from organic solvent(s)and/or water.

Each of the crystalline LNT forms A, B, C and D according to thisinvention is suitable for pharmaceutical and nutritional use. In thisregard, LNT, alone or in combination with other N-acetyllactosamineand/or fucose and/or sialic acid containing human milk oligosaccharides,is particularly effective in the education and/or maturation of theimmune system of neonatal infants, and have preventive effect againstsecondary infections following viral infections such as influenza. Theuse of LNT as prebiotic enhances the beneficial effects and efficiencyof probiotics, such as Lactobacillus and Bifidobacterium species, inpromoting the development of an early bifidogenic intestinal microbiotain infants, in reducing the risk of development or allergy and/or asthmain infants, in preventing and treating pathogenic infections in such asdiarrhoea in infants.

Also in accordance with this invention, a pharmaceutical composition isprovided comprising the crystalline LNT form A, B, C or D of theinvention, or their combination, as an active ingredient and one or morepharmaceutically acceptable carriers such as additives, adjuvants,excipients and diluents (water, gelatine, talc, sugars, starch, gumarabic, vegetable gums, vegetable oils, polyalkylene glycols, flavouringagents, preservatives, stabilizers, emulsifying agents, lubricants,colorants, fillers, wetting agents, etc.). Suitable carriers aredescribed in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field. The dosage form foradministration includes, for example, tablets, powders, granules, pills,suspensions, emulsions, infusions, capsules, syrups, injections,liquids, elixirs, extracts and tincture.

Also in accordance with this invention, the crystalline LNT form A, B, Cor D of the invention, or their combination, can be used for thepreparation of pharmaceutical compositions. Pharmaceutical compositionscan be manufactured in a conventional manner, e.g. as described in themost recent edition of Remington's Pharmaceutical Sciences, a standardreference text in the field.

Further in accordance with this invention, a nutritional formulation(such as food, drink or feed) is provided comprising the crystalline LNTform A, B, C or D of this invention, or their combination. Thenutritional formulation can also contain edible micronutrients, vitaminsand minerals as well. The amounts of such ingredient can vary dependingon whether the formulation is intended for use with normal, healthyinfants, children, adults or subjects having specialized needs (e.g.suffering from metabolic disorders). Micronutrients include for exampleedible oils, fats or fatty acids (such as coconut oil, soy-bean oil,monoglycerides, diglycerides, palm olein, sunflower oil, fish oil,linoleic acid, linolenic acid etc.), carbohydrates (such as glucose,fructose, sucrose, maltodextrin, starch, hydrolysed cornstarch, etc.)and proteins from casein, soy-bean, whey or skim milk, or hydrolysatesof these proteins, but protein from other source (either intact orhydrolysed) may be used as well. Vitamins may be chosen from the groupconsisting of vitamin A, B1, B2, B5, B6, B12, C, D, E, H, K, folic acid,inositol and nicotinic acid. The nutritional formulation can alsocontain the following minerals and trace elements: Ca, P, K, Na, Cl, Mg,Mn, Fe, Cu, Zn, Se, Cr or I.

In a preferred embodiment, the nutritional formulation is an infantformula, i.e. a foodstuff intended for use by infants during the first4-6 months of life and satisfying, by itself, the nutritionalrequirements of infants. It can contain one or more probioticBifidobacterium species, prebiotics such as fructooligosaccharides andgalactooligosaccharides, proteins from casein, soy-bean, whey or skimmilk, carbohydrates such as lactose, saccharose, maltodextrin, starch ormixtures thereof, lipids (e.g. palm olein, sunflower oil, safflower oil)and vitamins and minerals essential in a daily diet. The infant formulapreferably contains the crystalline LNT form A, B, C or D of theinvention, or their combination, in a total amount of 0.1-3.0 g/100 g offormula.

In another preferred embodiment, the nutritional formulation can be afood supplement containing the crystalline LNT form A, B, C or D of theinvention, or their combination. The food supplement can also containone or more probiotics in an amount sufficient to achieve the desiredeffect in an individual, preferably in children and adults. The foodsupplement can also contain vitamins, minerals, trace elements and othermicronutrients as well. The food supplement may be for example in theform of tablets, capsules, pastilles or a liquid. The supplement canalso contain conventional additives, such as binders, coatings,emulsifiers, solubilising agents, encapsulating agents, film formingagents, adsorbents, carriers, fillers, dispersing agents, wettingagents, jellifying agents, gel forming agents, etc. The daily dose ofLNT typically ranges from 0.1 to 3.0 g.

According to a more preferred embodiment, the food supplement is adigestive health functional food as the administration of LNT provides abeneficial effect on digestive health. Digestive health functional foodis a processed food used with intention enhance and preserve digestivehealth by crystalline LNT according to the present invention asphysiologically functional ingredient or component in forms of tablet,capsule, powder, etc. Different terms such as dietary supplement,nutraceutical, designed food, health product may also be used to referto functional food.

In a further embodiment, the crystalline LNT form A, B, C or D of theinvention, or their combination, is used for the preparation ofnutritional formulations, such as foods, drinks and feeds, preferablyinfant formulas, food supplements and digestive health functional food.The nutritional formulations can be prepared in a conventional manner.For example, it can be prepared by admixing micronutrient components inappropriate proportions, and then adding vitamins and minerals whileavoiding thermal degradation or decomposition of heat sensitive vitaminsby adding them after homogenization. Lipophilic vitamins can bedissolved in a fat source before mixing. A liquid mixture is formedusing water, whose temperature is preferably about 50-80° C. to helpdissolution or dispersal of the ingredients. The crystalline LNT form A,B, C or D, or their combination, can be suitably added at this stage.The resulting mixture is then homogenized by flash heating to about80-150° C. by means of steam injection, heat exchanger or autoclave.This thermal treatment also reduces significantly the bacterial load.The hot mixture is then cooled rapidly to about 60-80° C. If needed,further homogenization can be carried out at this temperature under highpressure of about 2-30 MPa. After cooling, heat sensitive constituentscan be added, and the pH and the solids content can be convenientlyadjusted. The resulting mixture is then dried in a conventional manner,such as by spray drying or freeze drying to powder. Probiotics can beadded at this point by dry-mixing.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not to be limiting 1.

EXAMPLES

XRPD investigation of the collected crystals was conducted with aPhilips PW 1830/PW1050 instrument in transmission geometry, using CuKαradiation made monochromatic by means of a graphite monochromator.D-spacings were calculated from the 2Θ values, based on a wavelength of1.54186 Å. As a general rule the 2Θ values have an error rate of±0.2.The recorded XRPD diagrams are shown in FIGS. 1 to 4.

Form A

LNT (1.0 g) was dissolved in water (5 ml). Abs. EtOH (4.5 ml) was addedat room temperature, resulting in a clear solution. Crystals wereobtained after stirring the solution for 18 hours at room temperature.The crystals were filtered off, washed with approx. 3 ml abs. EtOH anddried. Mp.: 226-228° C.

Form B

An aqueous solution containing LNT (17.5 g) was concentrated to Brix 48°and heated to 80° C. for full dissolution, then allowed to slowly coddown to room temperature. After 18 hours, a thick suspension wasobtained which was diluted with water (5 ml) to afford a nicelystirrable slurry. After 1 hour of stirring at room temperature thecrystals were filtered off, and the wet cake was dried on air for 9 daysto get a white solid (14.6 g).

Form C

I) An aqueous solution containing LNT (25 g) was concentrated to Brix37°, heated to 50° C. and methanol (50 ml) was added to the hotsolution. Seeding crystal of form B (100 mg) and a second portion ofMeOH (50 ml) were added. After 45 minutes a third portion of MeOH (50ml) was added. The obtained suspension was allowed to cool down andstirred at 25° C. for 2 hours. The solid was filtered off, washed with50 ml of MeOH and dried in vacuum oven for 17 hours at 60° C. and 30mbar (15.5 g). Mp.: 252-255° C.

II) LNT (9.7 g) dissolved in water (33 ml)) was heated to 60° C.Methanol (66 ml) was slowly added over 35 min. The solution was stirredat 60° C. and after 15 min spontaneous nucleation occurred. Thesuspension was stirred for 2 hours at 60° C. before being slowly cooledto 40° C. over 1.5 hours. The suspension was further stirred at 40° C.for 14 hours. The suspension was then allowed to cool to 25° C. andfurther stirred at this temperature for 2 hours. Crystals were filteredoff and washed with 10 ml of methanol. Wet crystals were dried at 50° C.and 50 mbar for 21 hours, giving a crystaline material having a watercontent of 9.4%, the XRPD pattern of which is identical with that of thesample obtained above.

III) An aqueous solution containing LNT (34 g) was concentrated to brix24°. At 35° C. a first portion of MeOH (150 ml) was added. The solutionwas seeded with polymorph C. Further MeOH (150 ml) was added in 25 mlportions over a period of 1 hour. The suspension was stirred at 35° C.for 3 hours, then heated up to 55° C., stirred at 55° C. for 6 hours,then cooled down to 35° C. and stirred at 35° C. for 4 hours. The solidwas filtered off, washed with 60 ml of MeOH and dried in vacuum oven at60° C. and 50 mbar for 5 hours to afford a crystalline material (29 g),which was identical, according to XRPD, with the sample obtained above,and had a water content of 9.1% (by Karl-Fisher titration).

Form D

I) An aqueous solution containing LNT (32 g) was concentrated to Brix24°, heated to 35° C. and MeOH (150 ml) was added to the warm solution.Seeding crystal form C (300 mg) and a second portion of MeOH (150 ml)was added in 25 ml portions over a period of 1 hour. The suspension wasstirred at 35° C. for 3 hours, at 55° C. for 6 hours, then was allowedto cool down to 30° C. and stirred for 4 hours at this temperature. Thesolid was filtered off, washed with 50 ml of MeOH and dried in oven at70° C. for 22 hours at atmospheric pressure (28.6 g). Mp.: 249-251° C.

II) LNT form C was further dried in a vacuum oven for 20 days at 60° C.and 20 mbar to give form D according to XRPD.

1.-8. (canceled)
 9. A crystalline form of LNT having an X-ray powderdiffraction pattern, characterized by peaks at two theta angle of19.25±0.2, 9.19±0.2 and 6.88±0.2 based on a measurement using CuKαradiation. 10.-17. (canceled)
 18. A pharmaceutical compositioncomprising the crystalline form of LNT of claim 9 and one or morepharmaceutically acceptable carriers.
 19. A nutritional formulationcomprising the crystalline form of LNT of claim
 9. 20. The nutritionalformulation of claim 19, wherein the nutritional formulation is aninfant formula.
 21. The nutritional formulation of claim 19, wherein thenutritional formulation is a food supplement.
 22. The nutritionalformulation of claim 21, wherein the food supplement is a digestivehealth functional food.
 23. (canceled)
 24. The crystalline form of LNnTof claim 9, wherein the X-ray powder diffraction pattern ischaracterized by peaks at two theta angles of 19.25±0.2, 9.19±0.2,6.88±0.2, 21.07±0.2 and 20.87±0.2, based on a measurement using CuKαradiation.
 25. The crystalline form of LNnT of claim 9, wherein theX-ray powder diffraction pattern is characterized by peaks at two thetaangles of 19.25±0.2, 9.19±0.2, 6.88±0.2, 21.07±0.2, 20.87±0.2 and19.94±0.2, based on a measurement using CuKα radiation.
 26. Thecrystalline form of LNnT of claim 9, wherein the X-ray powderdiffraction pattern is characterized by peaks at two theta angles of19.25, 9.19, 6.88, 21.07, 20.87, 19.94 and 19.66, based on a measurementusing CuKα radiation.
 27. The crystalline form of LNnT of claim 9,wherein the X-ray powder diffraction pattern is characterized by peaksat two theta angles of 19.25, 9.19, 6.88, 21.07, 20.87, 19.94, 19.66 and6.02, based on a measurement using CuKα radiation.
 28. The crystallineform of LNnT of claim 9, wherein the X-ray powder diffraction pattern ischaracterized by peaks at two theta angles of 19.25, 9.19, 6.88, 21.07,20.87, 19.94, 19.66, 6.02 and 22.30, based on a measurement using CuKαradiation.
 29. The crystalline form of LNnT of claim 9, wherein theX-ray powder diffraction pattern is characterized by peaks at two thetaangles of 19.25, 9.19, 6.88, 21.07, 20.87, 19.94, 19.66, 6.02, 22.30 and25.15, based on a measurement using CuKα radiation.
 30. The crystallineform of LNnT of claim 9, wherein the crystalline form of LNnT has anX-ray powder diffraction diagram as shown in FIG.
 2. 31. The crystallineform of LNnT of claim 9, wherein the crystalline form of LNnT has acapillary melting point from 239 to 243° C.